A comprehensive approach for establishment of the platform to analyze functions of KIAA proteins: generation and evaluation of anti-mKIAA antibodies

Since December 2001 we have been conducting a project to isolate and determine entire sequences of mouse KIAA cDNA clones which encode polypeptides corresponding to human KIAA proteins. The ultimate goal of this project is the elucidation of the functions of KIAA proteins. A critical step in this project is the generation of antibodies based on the cDNA sequence information. Although antibodies are the most optimal tools for biological analysis, the production and isolation of multiple recombinant proteins for an antigen is a rate-limiting step in antibody production. To address this problem, we established a system utilizing the in vitro recombination-assisted method and shotgun clones that were generated during the sequencing of mouse KIAA cDNAs (DNA Res. 2003, 10, 129-136). The authenticity of the expressed proteins was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Another critical step for antibody production is the evaluation of the antibodies. Thus, we also made efforts to develop a systematic approach for evaluation of the titer and the specificity of the antibodies. Using these systems, we have produced and evaluated more than 500 antibodies raised against mouse KIAA proteins to date. We are currently generating antibody arrays for analysis of protein expression profiles. We will verify protein-protein interactions using immunoprecipitation and tandem mass spectrometry analysis.     

A combination effect of epigallocatechin gallate, a major compound of green tea catechins, with antibiotics on Helicobacter pylori growth in vitro

Since green tea catechins are known to have antimicrobial activity against a variety of microorganisms, their possible effects on Helicobacter pylori in combination with antibiotics were examined. Fifty-six clinical isolates of H. pylori, including 19 isolates highly resistant to metronidazole (MTZ) and/or clarithromycin (CLR), were used to determine in vitro sensitivity to tea catechins. The MIC90 of both epigallocatechin gallate (EGCg) and epicatechin gallate (ECg) was 100 microg/ml. However, other tea catechins tested did not show any anti-H. pylori activity. Highly antibiotic-resistant clinical isolates showed a similar sensitivity to both EGCg and ECg. The kinetic study of antibacterial activity in liquid cultures revealed a relatively slow but strong activity on the growth of H. pylori. In combination with sub-MIC of amoxicillin (AMX), the antibacterial activity of AMX was significantly enhanced by the presence of EGCg. To estimate the general combination effect between EGCg and other antibiotics, such as MTZ and CLR, on the antibacterial activity against clinical isolates, the fraction inhibitory concentration (FIC) was determined by checkerboard study. The FIC indexes showed additive effects between EGCg and antibiotics tested. These results indicatethat EGCg may be a valuable therapeutic agent against H. pylori infection.     

Mechanism underlying the inner membrane retention of Escherichia coli lipoproteins caused by Lol avoidance signals

Escherichia coli lipoproteins are localized to either the inner or outer membrane depending on the residue at position 2. The inner membrane retention signal, Asp at position 2 in combination with certain residues at position 3, functions as a Lol avoidance signal, i.e. the signal inhibits the recognition of lipoproteins by LolCDE that releases lipoproteins from the inner membrane. To understand the role of the residue at position 2, outer membrane-specific lipoproteins with Cys at position 2 were subjected to chemical modification followed by the release reaction in reconstituted proteoliposomes. Sulfhydryl-specific introduction of nonprotein molecules or a negative charge to Cys did not inhibit the LolCDE-dependent release. In contrast, oxidation of Cys to cysteic acid resulted in generation of the Lol avoidance signal, indicating that the Lol avoidance signal requires a critical length of negative charge at the second residue. Furthermore, not only modification of the carboxylic acid of Asp at position 2 but also that of the amine of phosphatidylethanolamine abolished the Lol avoidance function. Based on these results, the Lol avoidance mechanism is discussed.     

Molecular identification of human glutamine- and ammonia-dependent NAD synthetases. Carbon-nitrogen hydrolase domain confers glutamine dependency

NAD synthetase catalyzes the final step in the biosynthesis of NAD. In the present study, we obtained cDNAs for two types of human NAD synthetase (referred as NADsyn1 and NADsyn2). Structural analysis revealed in both NADsyn1 and NADsyn2 a domain required for NAD synthesis from ammonia and in only NADsyn1 an additional carbon-nitrogen hydrolase domain shared with enzymes of the nitrilase family that cleave nitriles as well as amides to produce the corresponding acids and ammonia. Consistent with the domain structures, biochemical assays indicated (i) that both NADsyn1 and NADsyn2 have NAD synthetase activity, (ii) that NADsyn1 uses glutamine as well as ammonia as an amide donor, whereas NADsyn2 catalyzes only ammonia-dependent NAD synthesis, and (iii) that mutant NADsyn1 in which Cys-175 corresponding to the catalytic cysteine residue in nitrilases was replaced with Ser does not use glutamine. Kinetic studies suggested that glutamine and ammonia serve as physiological amide donors for NADsyn1 and NADsyn2, respectively. Both synthetases exerted catalytic activity in a multimeric form. In the mouse, NADsyn1 was seen to be abundantly expressed in the small intestine, liver, kidney, and testis but very weakly in the skeletal muscle and heart. In contrast, expression of NADsyn2 was observed in all tissues tested. Therefore, we conclude that humans have two types of NAD synthetase exhibiting different amide donor specificity and tissue distributions. The ammonia-dependent synthetase has not been found in eucaryotes until this study. Our results also indicate that the carbon-nitrogen hydrolase domain is the functional domain of NAD synthetase to make use of glutamine as an amide donor in NAD synthesis. Thus, glutamine-dependent NAD synthetase may be classified as a possible glutamine amidase in the nitrilase family. Our molecular identification of NAD synthetases may prove useful to learn more of mechanisms regulating cellular NAD metabolism.     

Insect cytokine growth-blocking peptide triggers a termination system of cellular immunity by inducing its binding protein

Growth-blocking peptide (GBP) is a 25-amino acid cytokine found in lepidopteran insects that possesses diverse biological activities such as stimulation of immune cells (plasmatocytes), cell proliferation, and larval growth regulation. We found another novel function of GBP that induces a hemolysis of another class of blood cells (oenocytoids). In the lysate of oenocytoids we identified a GBP-binding protein that shows a specific affinity for GBP. The characterization of purified GBP-binding protein and its cDNA demonstrated it as a 49.5-kDa novel protein with a C-terminal region displaying limited homology to several insect lipoproteins. Results of Northern and Western blotting indicated that the GBP-binding protein should be synthesized only in blood cells. Immunoelectron microscopic analyses confirmed that indirect immunoreactive signals were mostly localized in oenocytoids. Kinetic and biological analyses of interaction between GBP and the binding protein showed their strong binding was followed by clearance of GBP from hemolymph, thus indicating that this protein might function as an inhibitory factor against GBP. Based on these results, we propose that insect cytokine GBP shows multifunctions even in cellular immunity: it serves to stimulate immune cells and afterward silences its own action by inducing the binding protein through specific hemolysis.     

The biological activity and tissue distribution of 2',3'-dihydrophylloquinone in rats

2',3'-Dihydrophylloquinone (dihydro-K1) is a hydrogenated form of vitamin K1 (K1), which is produced during the hydrogenation of K1-rich plant oils. In this study, we found that dihydro-K1 counteracts the sodium warfarin-induced prolonged blood coagulation in rats. This indicates that dihydro-K1 functions as a cofactor in the posttranslational gamma-carboxylation of the vitamin K-dependent coagulation factors. It was also found that dihydro-K1 as well as K1 inhibits the decreasing effects of warfarin on the serum total osteocalcin level. In rats, dihydro-K1 is well absorbed and detected in the tissues of the brain, pancreas, kidney, testis, abdominal aorta, liver and femur. K1 is converted to menaquinone-4 (MK-4) in all the above-mentioned tissues, but dihydro-K1 is not. The unique characteristic of dihydro-K1 possessing vitamin K activity and not being converted to MK-4 would be useful in revealing the as yet undetermined physiological function of the conversion of K1 to MK-4.     

A novel method for study of protein folding kinetics by monitoring diffusion coefficient in time domain

Molecular diffusion process after the photo-induced electron injection to ferric cytochrome c (Fe(III) cyt c) in guanidine hydrochloride (GdnHCl) 3.5 M buffer solution is studied by the time-resolved transient grating technique. Circular dichroism studies have revealed that Fe(III) cyt c is unfolded under this condition but the reduced form, Fe(II) cyt c, is folded. Hence, this pulsed laser-induced reduction should initiate the folding process of cyt c. The observed transient grating signal shows prominent features, which have never been observed before. Based on several characteristic points, we concluded that the apparent diffusion coefficient (D) of Fe(II) cyt c after the reduction is time dependent, which must be associated with the protein folding dynamics. This time-dependent apparent D should reflect either the continuous time development of the hydrodynamic radius or population change of the unfolded and folded states during the folding dynamics. This is the first observation of the time-dependent apparent D during any chemical reaction, and this time-dependent measurement of D should be a unique and powerful way to study the protein folding kinetics from a viewpoint of the protein's shape or the protein-water intermolecular interaction.     

Neutralization of toxic heme by Plasmodium falciparum histidine-rich protein 2

Plasmodium falciparum histidine-rich protein 2 (PfHRP2) has been suggested to be an initiator of the polymerization of heme, which is produced as by-product on the digestion of hemoglobin, and a promoter of the H(2)O(2)-induced degradation of heme in food vacuoles of the malarial parasite. In this work, we have designed PfHRP2 model peptides, R18 and R27 (18 and 27 residues, respectively), and used them for optical and electron spin resonance spectroscopic measurements to confirm that the axial ligands of the heme-PfHRP2 complex are the nitrogenous donors derived from the imidazole moieties of histidine residues of PfHRP2. In addition, we revealed that the affinities of R18 and R27 for heme (K(d) = 2.21 x 10(-6) M and 0.71 x 10(-6) M, respectively) might be as high as that of PfHRP2 (K(d) = 0.94 x 10(-6) M). The R27 peptide can remove heme from membrane-intercalated heme and inhibit heme-induced hemolysis. Therefore, we suggest another function of PfHRP2: it may play an important role in the neutralization of toxic heme in the parasite cytoplasm and infected erythrocytes by removing heme from heme-bound membranes or reducing heme-induced hemolysis.